Rakesh Singal

EGFR Targeting of Solid Tumors

BACKGROUND Recent clinical trials suggest that epidermal growth factor receptor (EGFR)-targeted agents could benefit many patients with cancer. METHODS We review the current status of several EGFR-targeted therapies in cancer patients and address the efficacy of theses drugs as monotherapy or in combination with other drugs and/or treatments. RESULTS Cetuximab is the most widely studied anti-EGFR monoclonal antibody. Other monoclonal antibody agents under investigation are panitumumab, matuzumab, MDX-447, nimutozumab, and mAb806. Extensive research has also evaluated the efficacy of EGFR tyrosine kinase inhibitors such as erlotinib, gefitinib, EKB-569, lapatinib (GW572016), PKI-166, and canertinib (CI-1033). All of these agents have been studied for the treatment of colorectal, lung, breast, pancreatic, renal, head and neck, gynecologic, and prostate cancer. Currently, cetuximab and panitumumab are FDA approved for the treatment of metastatic colorectal cancer. Additionally, cetuximab is approved for head and neck cancer. Erlotinib is FDA approved for advanced/metastatic lung cancer. Erlotinib in combination with gemcitabine is approved for advanced/metastatic pancreatic cancer treatment. CONCLUSIONS EGFR-targeted agents have already shown utility in different scenarios. Researchers are continuously investigating additional cancer types and combined treatment modalities that could also benefit from the use of EGFR-targeted agents. Careful patient selection through the identification of specific biologic markers, such as gene expression, genomic polymorphism, and posttranslational modifications of EGFR downstream effectors, most likely will contribute to the successful use of these agents.

Chromatin immunoprecipitation assay.

Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.

Aberrant promoter methylation of multiple genes in malignant ovarian tumors and in ovarian tumors with low malignant potential

Methylation‐mediated suppression of detoxification, DNA repair, and tumor suppressor genes has been implicated in cancer development and progression. Studies also have indicated that concordant methylation of multiple genes (methylator phenotypes), rather than a single gene, may predict cancer prognosis. The current study was designed to determine whether a methylator phenotype exists in ovarian cancer, whether methylation frequencies differ between malignant ovarian tumors and ovarian tumors with low malignant potential (LMP or borderline), and whether methylation of multiple genes affects patient survival.

Soluble CD44 Is a Potential Marker for the Early Detection of Head and Neck Cancer

Introduction: Head and neck squamous cell carcinoma (HNSCC) is a devastating and deadly disease, largely because it is diagnosed in late stage. Cure rates, currently at 50%, could increase to >80% with early detection. In this study, we evaluate soluble CD44 (solCD44) as an early detection tool for HNSCC by determining whether it reliably distinguishes HNSCC from benign disease of the upper aerodigestive tract. Methods: We carried out the solCD44 ELISA on oral rinses from 102 patients with HNSCC and 69 control patients with benign diseases of upper aerodigestive tract to determine the sensitivity and specificity of the test for differentiating HNSCC from benign disease. Furthermore, we did a pilot study using methylation-specific PCR primers on oral rinses from 11 HNSCC patients with low solCD44 levels and 10 benign disease controls. Results: Mean salivary solCD44 levels were 24.4 ± 32.0 ng/mL for HNSCC patients (range, 0.99-201 ng/mL) and 9.9 ± 16.1 ng/mL (range, 0.73-124 ng/mL) for the patients with benign disease (P < 0.0001). Depending on cutoff point and HNSCC site, sensitivity ranged from 62% to 70% and specificity ranged from 75% to 88%. Nine of 11 HNSCC and 0 of 10 controls with low solCD44 levels showed hypermethylation of the CD44 promoter. Conclusions: SolCD44 is elevated in the majority of HNSCC and distinguishes cancer from benign disease with high specificity. Whereas the solCD44 test lacks sensitivity by itself, methylation status of the CD44 gene seems to complement the solCD44 test. Our pilot data indicate that, together, these markers will detect HNSCC with very high sensitivity and specificity. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1348–55)

Transcriptional regulation by the estrogen receptor of antioxidative stress enzymes and its functional implications

We previously reported that antiestrogen-liganded estrogen receptor β (ERβ) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Our studies also indicate that upregulation of QR, either by overexpression or induction by tamoxifen, can protect breast cells against oxidative DNA damage caused by estrogen metabolites. We now report on the upregulation of glutathione S-transferases Pi (GST-Pi) and gamma-glutamylcysteine synthetase heavy subunit (GCSh) expression by antiestrogens. Studies indicate the regulation of GST-Pi and GCSh transcriptional activity by ER. While ER regulation is mediated by an electrophile response element (EpRE), we identified mechanistic differences in the involvement of other transcription factors. Regardless of these differences, ERβ-mediated regulation of GST-Pi and GCSh point towards an important role for ERβ in cellular protection against oxidative stress. A protective role is supported by our observation of inhibition of estrogen-induced DNA damage upon upregulation of GST-Pi and GCSh expression.

Epigenetics in Prostate Cancer

Prostate cancer (PC) is the most commonly diagnosed nonskin malignancy and the second most common cause of cancer death among men in the United States. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequences. Two common epigenetic mechanisms, DNA methylation and histone modification, have demonstrated critical roles in prostate cancer growth and metastasis. DNA hypermethylation of cytosine-guanine (CpG) rich sequence islands within gene promoter regions is widespread during neoplastic transformation of prostate cells, suggesting that treatment-induced restoration of a “normal” epigenome could be clinically beneficial. Histone modification leads to altered tumor gene function by changing chromosome structure and the level of gene transcription. The reversibility of epigenetic aberrations and restoration of tumor suppression gene function have made them attractive targets for prostate cancer treatment with modulators that demethylate DNA and inhibit histone deacetylases.

Characterization of CD44v3-containing isoforms in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease that is only cured 50% of the time. A better understanding of the molecular mechanisms involved in HNSCC progression may lead to earlier detection and improved cure rates. CD44 is a ubiquitous transmembrane glycoprotein comprising a family of alternatively spliced isoforms involved in cell migration and cell proliferation. CD44 isoforms containing the variant 3 (v3) exon include a growth factor binding site and may be involved in tumor progression. To characterize CD44v3-containing isoforms expression in HNSCC we purified RNA from four HNSCC cell lines and performed RTPCR using junction primer strategies followed by gel elecrophoresis. Cloning and sequencing of HNSCC cell line PCR products revealed two isoforms. One of these, CD44v3-10, has been previously described. The other isoform, CD44v3, has not been characterized in HNSCC tissues. To further study this isoform, we purified RNA from 19 HNSCC tissues, 7 normal margin tissues and 5 true normal tissues. Following reversetranscription, we performed quantitative PCR using junction primers specific for CD44v3. Results show that HNSCC tumor tissues expressed mean CD44v3 levels that were elevated 4.5 times more than true normal tissues (p

Serum Free Circulating DNA Is a Useful Biomarker to Distinguish Benign versus Malignant Prostate Disease

Background: Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a “representative” patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting. Methods: The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene. Results: Patients with PSA ≤ 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA ≤180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA. Conclusions: Our results indicate that fcDNA may improve the specificity of prostate cancer screening. Impact: Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies. Cancer Epidemiol Biomarkers Prev; 19(8); 1984–91. ©2010 AACR.

Phase I/II Study of Azacitidine, Docetaxel, and Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer Previously Treated With Docetaxel-Based Therapy

INTRODUCTION Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. PATIENTS AND METHODS Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 + 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. RESULTS In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6 with growth factor support. Prostate-specific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. CONCLUSION The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients.

Epigenetic targets in the diagnosis and treatment of prostate cancer

Prostate cancer (PC) is one of leading cause of cancer related deaths in men. Various aspects of cancer epigenetics are rapidly evolving and the role of 2 major epigenetic changes including DNA methylation and histone modifications in prostate cancer is being studied widely. The epigenetic changes are early event in the cancer development and are reversible. Novel epigenetic markers are being studied, which have the potential as sensitive diagnostic and prognostic marker. Variety of drugs targeting epigenetic changes are being studied, which can be effective individually or in combination with other conventional drugs in PC treatment. In this review, we discuss epigenetic changes associated with PC and their potential diagnostic and therapeutic applications including future areas of research.